Devices and methods of cell capture and analysis

ABSTRACT

The present invention provides a device for isolating target biomolecules or cells from samples, particularly biological samples. In particular, the device comprises a loading mixture, which contains the biological sample and a first binding entity that specifically binds to the target biomolecule or target cell; and a micro-channel coated with a second binding entity that binds directly or indirectly to the first binding entity. Methods of capturing, detecting, and/or evaluating target biomolecules or target cells (e.g. cancer cells) in biological samples are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/812,498, filed on Jul. 29, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/730,738, filed on Mar. 24, 2010 (now U.S. Pat.No. 9,128,082, issued on Sep. 8, 2015), which claims the benefit ofpriority to U.S. Provisional Patent Application No.: 61/298,871, filedon Jan. 27, 2010, U.S. Provisional Patent Application No. 61/235,615,filed on Aug. 20, 2009 and U.S. Provisional Patent Application No.61/163,009, filed on Mar. 24, 2009, each of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to micro-channel devices for capturingtargets, such as cells and molecules of interest from solutions, as wellas to post-capture analysis of circulating cells. In certainembodiments, the present invention relates to methods and devices forcapturing target cells (e.g. circulating tumor cells) from physiologicalfluids, and analyses thereof.

BACKGROUND OF THE INVENTION

Isolation of target cells or molecules from heterogeneous samplesremains a prominent interest for research applications as well asmedical applications, such as diagnostics and therapeutics. Inparticular, separation of rare cell types from physiological tissues andbodily fluids obviates the need to obtain large tissue samples andavoids the risks associated with the procedures required to obtain suchsamples. For example, isolation of fetal cells from maternal bloodsamples for genetic testing avoids the risks associated withaminocentesis or chronic villus sampling. Isolation of circulating tumorcells from a patient would allow the clinician to evaluate the cancerand monitor pathological changes in the patient's tumor, as well asevaluate the efficacy of any on-going drug treatments without conductinginvasive biopsy procedures.

Current methods for separating biological molecules and/or cells fromheterogeneous samples typically entail the use of a high affinitybinding partner (e.g. an antibody or antigen) coupled to a solidsupport. The heterogeneous sample is passed over the solid support andthe target biological molecules or cells of interest are bound by thebinding partner and retained on the solid support. The bound moleculesor cells of interest can be subsequently analyzed for the presence ofmolecular genomic and proteomic information.

These current approaches suffer from several technical difficulties, oneof which is the problem of non-specific binding. To minimizenon-specific binding, one or more washing steps is required to removeother molecules and/or cells that are bound to the solid support orbinding partner. In addition, the subsequent in situ analysis of cellson the channel by staining and hybridization procedures may subject thecells to harsh and denaturing conditions. These washing and analysisprocedures can compromise the initial capture of the desired molecule orcell by subjecting the binding partner to conditions that may cause thebinding partner to degrade, lose some of its conformational structure,or become detached from the solid support.

Further still, existing methods for analyzing circulating cells (e.g.,as captured from a patient sample) for malignancy, such as stainingcells for cytokeratin (CK), have limitations as markers for identifyingand/or evaluating circulating tumor cells.

Thus, there is a need in the art for additional methods and devices forisolating biological molecules and/or cells of interest from samples, aswell as methods for subsequent analysis of captured targets, such asanalysis of captured, circulating tumor cells.

SUMMARY OF THE INVENTION

The present invention provides devices and methods for capturing and/oranalyzing biological targets from fluid samples. In various embodiments,the invention provides methods for capturing circulating tumor cellsfrom biological samples, for the evaluation of a cancer patient'sdisease. In these and other embodiments, the invention provides methodsfor identifying and/or evaluating circulating cells for malignancywithout or independent of CK status.

In one aspect, the invention provides a method for capturing biologicaltargets from solution. In this aspect, the present invention is based,in part, on the discovery that pre-labeling or pre-mixing a samplecontaining a target (e.g., a cell) of interest with a binding partnerthat specifically binds to the cell enhances the capture of such targetsin a micro-channel device.

In certain embodiments, the device comprises a micro-channel and aloading mixture. The micro-channel may comprise a population of postsdistributed on the surface of the micro-channel in random pattern. Theloading mixture may comprise a biological sample suspected of containinga target, such as a target cell, and also comprises a first bindingentity. The first binding entity specifically binds to the target (e.g.,a target entity on a target cell). The surface of the micro-channel iscoated with a second binding entity that specifically binds, directly orindirectly, to the first binding entity. In some embodiments, theloading mixture further comprises a third binding entity conjugated to adetectable or capturable entity. For example, the first binding entitymay be a primary antibody, the third binding entity may be a secondaryantibody that specifically binds to the primary antibody, and the secondbinding entity specifically binds directly or indirectly to thesecondary antibody. In one embodiment, the third binding entity is abiotinylated secondary antibody that specifically binds to the firstbinding entity and the second binding entity is avidin. The secondaryantibody may be intact antibody or any antibody fragment such as Fab′2,Fab′ or Fab. In addition this may include any of the geneticallyengineered or expressed forms of antibody fragment such as single chainFab fragment or single chain variable fragment.

In another aspect, the present invention provides a method for capturingand/or detecting a target cell in a biological sample, including rarecell populations as described herein. In one embodiment, the methodcomprises contacting a biological sample with a first binding entity toform a pre-loading mixture, wherein the first binding entityspecifically binds to a target entity on the surface of the target cell;passing the pre-loading mixture through a micro-channel, wherein thesurface of the micro-channel is coated with a second binding entitycapable of specifically binding to the first binding entity; anddetecting the presence of the target cell on the surface of themicro-channel. The biological sample can be a physiological or bodilyfluid or tissue, such as blood, plasma, serum, bone marrow, semen,vaginal secretions, urine, amniotic fluid, cerebral spinal fluid,synovial fluid, fine needle aspirates (FNAs) or biopsy tissue sample. Incertain embodiments, the target cell is rare and present at a low ratioin the biological sample. Examples of target cells that are rare in thebiological samples (e.g., blood) include circulating tumor cells (CTCs),cells that are in early stages of a disease state such as Stage 1 oftumorigenesis, as well as viral-, bacterial-, or fungal-infected cells.

In certain embodiments, the target cell is a cancer cell (e.g., acirculating tumor cell), such as a breast cancer cell, a prostate cancercell, a colorectal cancer cell, a lung cancer cell, a pancreatic cancercell, an ovarian cancer cell, a bladder cancer cell, an endometrine oruterine cancer cell, a cervical cancer cell, a liver cancer cell, arenal cancer cell, a thyroid cancer cell, a bone cancer cell, a lymphomacell, a melanoma cell and a non-melanoma skin cancer cell. The tumor maybe an epithelial tumor. In such embodiments, the first binding entitycan be an antibody that specifically binds to circulating epithelialcells. In one embodiment, the first binding entity is an epithelial celladhesion molecule antibody (e.g., EpCAM). In these and otherembodiments, the first binding entity is a biotinylated-antibody and thesecond binding entity is avidin. In various embodiments, the inventioninvolves antibody cocktails as the first binding entity, so as tocapture circulating tumor cells exhibiting a range of epithelial,mesenchymal, stem or progenitor cell characteristics.

In another embodiment of the invention, the pre-loading mixture furthercomprises a third binding entity. In such embodiments, the first bindingentity may be a primary antibody, the third binding entity may be asecondary antibody conjugated to a detectable or capturable entity andthe secondary antibody specifically binds to the first binding entity. Asecond binding entity specifically binds to the third binding entity viathe capturable moiety. In certain embodiments, the third binding entityis a biotinylated secondary antibody that specifically binds to thefirst binding entity, and the second binding entity is avidin.

In some embodiments, the method further comprises, after cell capture,cross-linking the target cell bound to the surface of the micro-channel.Cross-linking reagents include protein cross-linking reagents, such as ahydrophilic homobifunctional NHS crosslinking reagent. In certainembodiments, the captured cells can be subjected to further analysis inthe micro-channel or outside the channel post capture.

In another aspect, the invention provides a method for post-captureanalysis of circulating cells, and in particular, to examine or evaluatethe circulating cells for malignancy. Generally, the invention in thisaspect involves evaluating captured cells for aneuploidy, optionallywith evaluation of other markers of malignancy, including mutations. Themethod generally does not involve determining, or is independent of,cytokeratin expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of a micro-channel devicecomprising a post-containing collection region in the micro-channel.

FIG. 2 is a schematic depicting capture of a circulating tumor cell(CTC) in a micro-channel device that has been coated with an antibodyspecific to an antigen on the CTC. B designates biotin.

FIG. 3 is a schematic depicting capture of a circulating tumor cell(CTC) in a micro-channel device where the CTC has been pre-labeled withan antibody specific to a CTC antigen and the micro-channel device hasbeen coated with a protein capable of binding the cell-specificantibody. B designated biotin.

FIG. 4 is a graph showing percentage of T24 EpCAM positive cellscaptured on either a micro-channel coated with EpCAM antibodies (EpCAMchannel) or a micro-channel coated with streptavidin (Strep channel) atdifferent flow rates. In the case of the Strep channel, the T24 cellswere pre-labeled with a biotinylated EpCAM antibody prior to passageover the Strep channel.

FIG. 5 is a graph showing the percentage of T24 cells pre-labeled withbiotinylated EpCAM that were captured on a micro-channel coated withstreptavidin in the presence of different concentrations of excessbiotinylated EpCAM antibody. A sample of 250 μL containing approximately200 cells was applied to the channel.

FIG. 6 is a graph showing the percentage of T24 cells pre-labeled withbiotinylated EpCAM that were captured on a micro-channel coated withstreptavidin in the presence of different concentrations of excessbiotinylated EpCAM antibody. A sample of 2 mL containing approximately200 cells was applied to the channel.

FIG. 7 is a graph showing the dilution of the EpCAM capture antibodythat is coated onto the micro-channel compared to the capture of T24cells as a function of the same dilution mixture used to pre-label cellsprior to application onto the micro-channel.

FIG. 8 is a graph depicting the percentage of T24 or SKOV cells capturedon a streptavidin-coated micro-channel when pre-labeled with eitherbiotinylated Trop-1 antibody alone or in combination with biotinylatedTrop-2 antibody.

FIG. 9 is a graph depicting the capture of MDA-ND-231 cells on astreptavidin-coated micro-channel when pre-labeled with eitherbiotinylated anti-EpCAM antibody alone or in combination with a mixtureof biotinylated capture antibodies.

FIG. 10 is a graph depicting the percentage of captured SKOV cells on astreptavidin-coated micro-channel when pre-labeled with biotinylatedprimary antibody or a combination of non-biotinylated primary antibodyand biotinylated secondary antibody.

FIG. 11 shows a series of photomicrographs of cells captured in a coatedmicro-channel that were subsequently subjected to washes with a viscoussolution at different flow rates (20, 50, and 100 μL/min).

FIG. 12 shows a series of photomicrographs of cells captured in a coatedmicro-channel. The cells were exposed to a homobifunctional NHS proteincross-linking reagent prior to being subjected to washes with a viscoussolution at different flow rates (20, 50, and 100 μL/min).

FIG. 13 is a graph showing the percentage of captured cells on a coatedmicro-channel in the absence or presence of a NHS protein cross-linkingreagent.

FIG. 14 is a graph showing the percentage of captured bladder cancercells on a coated micro-channel when using EpCAM only as the captureantibody compared to using a mixture of antibodies. The graph also showsthe staining of the cell types with anti-cytokeratin and anti-vimentinantibodies.

FIG. 15A is a graph showing the capture of SKOV cells by EpCAM antibodycompared to capture by an antibody mixture.

FIG. 15B shows the staining of SKOV cells after incubation with EpCAMantibody or antibody mixture and detected with fluorescently labeledsecondary anti-mouse antibody. FACS analysis of the same cells shows thenumber of surface antigens labeled with labeled secondary anti-mouseantibody.

FIG. 16 is an image of SKOV cells spiked into blood and captured on amicrochannel using a primary antibody mixture and biotinylated secondaryanti-mouse antibody. Cells were stained on the channel withfluorescently labeled neutravidin which tightly binds biotin. Imageshows SKOV cell stained green with NeutrAvidin and nearby white bloodcells that did not stain with neutravidin but stained only with DAPI todetect the nucleus.

FIG. 17 shows the recovery of SKBr3 in a blood sample spiked withvarying numbers of SKBr3 cells. The results show that the percentcapture is independent of the cell input.

DETAILED DESCRIPTION

The present invention provides devices and methods for capturing and/oranalyzing biological targets from fluid samples. In various embodiments,the invention provides methods for capturing circulating tumor cellsfrom biological samples, for the evaluation of a cancer patient'sdisease. In these and other embodiments, the invention provides methodsfor identifying and/or evaluating circulating cells for malignancywithout or independent of cytokeratin expression.

In one aspect, the invention is based, in part, on the discovery thatpre-labeling or pre-mixing a sample containing a target of interest witha binding partner that specifically binds to the target allows, e.g.,enhances the capture of such targets in a micro-channel device, such asa microchannel device described herein. This approach also providesflexibility in the type and nature of primary antibodies that may beused to label cellular antigens. Accordingly, the present inventionprovides a novel device and method for separating biomolecules or cellsof interest from samples, particularly biological samples. In oneembodiment, the device comprises a micro-channel and a loading mixture.The micro-channel may comprise a population of posts distributed on thesurface of the micro-channel in random pattern. The loading mixture maycomprise a biological sample suspected of containing a target cell and afirst binding entity, wherein the first binding entity specificallybinds to a target entity on the target cell. The surface of themicro-channel is coated with a second binding entity that specificallybinds to the first binding entity, either directly or indirectly.

Any suitable micro-channel device may be used in connection with thepresent invention. In some embodiments, the micro-channel devicecomprises a plurality of pre-determined flow paths. In some embodiments,the micro-channel device comprises posts or obstacles arranged in arandom pattern or a regular or repeat pattern. In some embodiments, themicro-channel device comprises regions providing streamlined flow orrandom non-streamlined flow for any fluid passing through.

The micro-channel device may be a random-flow device for separatingbiomolecules or cells as described in detail in U.S. PublishedApplication No. 2006/0160243, which is hereby incorporated by referencein its entirety. Such devices can be modified as described herein foruse in connection with the invention. In general, the random-flowmicro-channel device includes a substrate or support that has a flowpath defined therein that includes at least one micro-channel having acollection region, which flow path is linked to a sample inlet and aliquid outlet. In some embodiments, the flow path may include severalmicro-channels, arranged in series, each of which has one suchcollection region. Alternatively, a random flow micro-channel may havemore than one collection region, arranged in series, and there may alsohave more than one inlet and more than one outlet. One particularembodiment of the random flow micro-channel device is described inExample 1 and illustrated in FIG. 1.

The collection region of the random flow micro-channel can contain aplurality of upstanding posts that are aligned transverse to the liquidflow path and arranged in an irregular, random pattern across the entirewidth of the collection region portion of the flow channel. In oneembodiment, the pattern of the posts is such that there can be nostraight-line flow through the collection region and/or that streamlinedflow streams are disrupted, assuring there is good contact between theliquid being caused to flow along the flow path and the surfaces of theposts. The posts in general are integral with the flat base of thecollection region and extend perpendicular thereto, presenting surfacesthat are vertical relative to a horizontal path of liquid being causedto flow through the flow channel of the substrate or support.

The placement and shape of the posts in the patterned post collectionregion can be engineered for optimal fluid dynamics and enhancement ofcapture of target cells through their specific surface characteristics.Very generally, in most instances, the preferred shape of the horizontalcross-section of the transverse fixed posts avoids sharp angles whichmight promote nonspecific binding to the transverse surfaces of theposts. The posts have rectilinear exterior surfaces and preferably haveeither a generally circular cross sectional shape or regular polygonalof 6 or more sides. Alternative shapes that might be used are atear-drop shape where the tip is at the downstream end and shallowlycurved, or oval shape; however, should more impact be desired, a squareshape could be used. In one embodiment, the pattern of the posts shouldcreate a flow pattern in the liquid stream which enhances the capture oftarget cells by the second binding entity attached to the surfaces ofthe posts, the base and the facing surface. To achieve this end, theposts, e.g., should be of different sizes and be arranged in a setrandom pattern. A random pattern of posts of different cross sectionalsizes, e.g. circular cross section posts of at least about 3 or 4different sizes, about 70 to about 130 microns in diameter, in acollection region about 100 microns high where the minimum separationspacing between posts is 50 to 70 μm and preferably about 60 μm.

In some embodiments, the cross sectional area of the posts, which allhave sidewalls formed by parallel lines which are perpendicular to thebase, is such that they occupy between about 10 to 40% or about 15 to25% of the volume of the collection region. Preferably the post patternwill be such that they occupy about 20% of the volume of the collectionregion, leaving a void volume for liquid flow of about 80%. The postsare substantially spaced apart from one another, e.g. by at least about60 microns, and posts of different sizes are preferably located upstreamand downstream of one another. Smaller posts may create eddy regionsdownstream of larger posts, and as a result of the flow pattern that isgenerated, the surfaces in the vicinity may show particulareffectiveness in capturing target cells.

Generally, the substrate component of the micro-channel device can bemade from any suitable laboratory-acceptable material, such as silicon,fused silica, glass and polymeric materials. It may be desirable to usea material that is optically transparent, particularly when a diagnosisfunction is desired to be optionally employed. In its simplestembodiment, the substrate carrying the fabricated micro-channel issealed with a plate having a flat surface that will abut the facingsurface of the substrate. Such plate may be fabricated from the samematerial or may simply be a cover plate made of glass. Suitable plasticswhich may be used include polydimethylsiloxane (PDMS),polymethylmethacrylate (PMMA), polycarbonate, polystyrene, polyethyleneteraphthalate, as well as other polymeric resins well known foracceptable laboratory material usage. Such patterned substrates may befabricated using any convenient method such as those selected from amongconventional molding and casting techniques.

Substrates may be conveniently fabricated from polymeric materials usinga master or negative mold structure, which can be created in a thicknegative photoresist, using optical lithography, as well known in thisart. For instance, the construction layer can be formed from a mixtureof commercially available, standard grade epoxy resin (EPON SU-8)photoresist and hardener (SU-82025), which may be spun onto siliconwafer substrates at 2000 rpm to provide, for example, a 40 or 50 μmthick film of such photoresist. The thickness determines the height ofthe flow path in the collection region. The film is subjected topre-exposure baking for 3 minutes at 60° C. and then 7 minutes at 95° C.on a precisely level hot plate to assure even thickness throughout, andthe resultant samples are cooled to room temperature. A Karl SussContact Mask Aligner is used to expose a film with the desired patternfor the flow path in the ultimate device. The film is then post-baked at65° C. for 2 minutes and then at 95° C. for 5 minutes before it isdeveloped in a commercial SU-8 developer for 5 minutes, with lightstirring being applied during developing. This creates a negativepattern mold in the epoxy resin photoresist that is then used as amolding master for replication of patterned post substrates in PDMS orother suitable polymeric resin. The layout and the dimensions of themicro-channel and of patterned posts in the collection region aredetermined by the mask used in exposure step of the fabrication of themaster mold. The depth of the micro-channel is controlled by thethickness of the SU-8 layer of the master mold, which is determined byspin-coating conditions.

The invention further involves a loading mixture that comprises abiological sample suspected of containing a target (e.g., a targetcell), and also comprises a first binding entity. The biological samplecan include, but is not limited to, a physiological or bodily fluid ortissue or a cell mixture isolated from a biological sample. For example,the biological sample can include, without limitation, blood, plasma,serum, semen, vaginal secretions, urine, saliva, amniotic fluid,cerebral spinal fluid, synovial fluid, a fine needle aspirate (FNA), anda biopsy tissue sample. A target cell can be any cell comprising adetectable surface antigen, such as a cancer cell, stem cell, fetalcell, a viral-, a bacterial- or a fungal-infected cell. In someembodiments, the target cell is a cancer cell. In certain embodiments,the target cell is rare and is present at a low ratio in the biologicalsample, or expresses a very low level of a particular antigen ofinterest. Examples of target cells that are rare in the biologicalsamples include circulating tumor cells (CTCs), cells that are in earlystages of a disease state such as cells at Stage 1 of tumorigenesis,early viral-, bacterial, or fungal-infections.

Preferably, the first binding entity specifically binds to a targetentity on the target cell. The first binding entity can include, but isnot limited to, an antibody, an antigen, an aptamer, a nucleic acid(e.g. DNA and RNA), a protein (e.g. receptor, enzyme, enzyme inhibitor,enzyme substrate, ligand), a peptide, a lectin, a fatty acid or lipidand a polysaccharide. In one embodiment, the first binding entity is anantibody. In another embodiment, the first binding entity comprises abinding entity mixture having at least a first antibody and a secondantibody, and wherein the first antibody specifically binds to a firstepitope of the target entity and the second antibody specifically bindsto a second epitope of the target entity. The first binding entity cancomprise a mixture of antibodies or binding entities directed to the oneor more target antigens on the cell, or one or more epitopes of thetarget antigen, or a combination thereof. As used herein the term“epitope” can refer to a binding region on a singular antigen or abinding region on a second antigen. By way of example, in someembodiments, the first antibody binds to a first epitope on a firstantigen and the second antibody binds to a second epitope on the firstantigen. In other embodiments, the first antibody binds to a firstepitope on a first antigen and the second antibody binds to a secondepitope on a second antigen. In certain embodiments, the antibodies maybe conjugated to a tag molecule including, but not limited to biotin,digoxigenin, FLAG epitope, or polyhistidine.

In another embodiment of the invention, the loading mixture furthercomprises a third binding entity conjugated to a detectable orcapturable entity. For example, the first binding entity may be aprimary antibody or ligand, the third binding entity is a secondaryantibody or ligand that specifically binds to the first binding entity,and the second binding entity specifically binds to the third bindingentity. A primary antibody can include a monoclonal antibody, apolyclonal antibody, or partially purified antibodies. The secondaryantibody can be an antibody that binds to the constant region of theprimary antibody. By way of example, if the primary antibody is a mouseantibody, the secondary antibody may be an anti-mouse antibody. Thedetectable or capturable entity conjugated to the secondary antibody canbe a tag including, but not limited to, biotin, digoxigenin, FLAGepitope, or polyhistidine. In one embodiment, the loading mixturefurther comprises a third binding entity, wherein the first bindingentity is a primary antibody, the third binding entity is a biotinylatedsecondary antibody that specifically binds to the first binding entityand the second binding entity is avidin. As used herein, the term“avidin” includes any expressed or engineered form of the avidinbiotin-binding molecule, such as streptavidin, neutravidin and the like.

The surface of the micro-channel of the device is coated with a secondbinding entity that specifically binds to the first binding entity. Thesecond binding entity can be an antibody, an antigen, an aptamer, anucleic acid (e.g. DNA and RNA), a protein (e.g. receptor, enzyme,enzyme inhibitor, enzyme substrate, ligand), a peptide, a lectin, afatty acid or a lipid, and/or a polysaccharide. The second bindingentity may be the same type of molecule as the first binding entity(e.g. antibody-antibody or nucleic acid-nucleic acid) or it may be adifferent type of molecule than the first binding entity (e.g. nucleicacid-protein). The second binding entity can directly bind to the firstbinding entity or it can indirectly bind to the first binding entitythrough a tag molecule. For instance, if the first binding entity is abiotinylated primary antibody, the second binding entity can be avidin.In one embodiment, the second binding entity is avidin. In someembodiments, the loading mixture can comprise both a first bindingentity and a third binding entity, wherein the first binding entitybinds to a target entity (e.g., on the target cell) and the thirdbinding entity specifically binds to the first binding entity. In suchembodiments, the second binding entity specifically binds to the thirdbinding entity either directly or indirectly through a detectableentity. By way of example, if the first binding entity is a mouseprimary antibody and the third binding entity is an anti-mouse antibodyconjugated to digoxigenin, then the second binding entity can be ananti-digoxigenin antibody.

The polymeric surface of the micro-channel and/or the patterned post orobstacle region comprised therein can be derivatized in various ways toenable the attachment of the second binding entity onto all thesurfaces. For example, after plasma treatment and closure of themicro-channel-carrying substrate, a 1 to 50 volume % solution of anaminofunctional silane (e.g. a 3% solution of Dow Corning Z-6020), or athio-functional silane, in ethanol may be injected into themicro-channel to fill the collection region between the sample inlet andsample outlet regions, and the flooded micro-channel can then be left toincubate for 30 minutes at room temperature. Derivitization can beperformed on a non-fully cured polymer, such as PDMS, before the closureof the micro-channel region with a plate. In such case, an alternativeis to slightly undercure the PDMS substrate and then complete the curingafter affixing the seal plate and treating with the substituted silaneor other functionalizing reagent. For example, a final heating step ofabout 90 minutes at about 50 to 90° C. might be used to complete thecuring after treating with the Z-6020. Alternatively, one or two days atroom temperature would also complete the curing. Such derivatizationtreatment can also be performed before the closure of the micro-channelregion because derivatization of the facing flat surface is of no realconsequence. The flow path is then purged with ethanol, and themicro-channel is ready for attachment of the second binding entity.

Second binding entities can be directly or indirectly immobilized uponthe surfaces of the posts, obstacles, and/or the micro-channel, and thesurfaces can be pre-treated and/or coated to facilitate attachment. Insome embodiments, indirect immobilization is preferred and contemplatesthe employment of an intermediate agent or substance that is firstlinked to the post or surface. It may be desired to use coupling pairsto link to the intermediate agent. For example, avidin, or an antibodydirected against another species antibody, might be attached to theintermediate agent, such as a NHS/maleimide heterobifunctional linker,which would thereafter couple to a biotinylated antibody or to anantibody of such other species.

Flow through the devices of the invention can be achieved by anysuitable means, with or without exterior force. In one embodiment, flowthrough the devices of the invention is achieved by pumping, e.g. usinga syringe pump or the like, or by vacuum that would draw liquid throughfrom a reservoir at an inlet well provided by a large diameter inlethole. Preferably such a well is included which has a capacity to holdabout 50 μl to about 500 μl of liquid sample. In one embodiment, thedesign of the flow channel is such that, at flow rates through thedevice within a reasonable range (e.g. by injection of sample using asyringe pump or equivalent device, such as a Biocept syringe pump, or astandard Harvard Apparatus infusion syringe pump or other commerciallyavailable syringe pump) to create a flow in the collection region at arate of about 0.01 to 100 mm per second, there is substantial disruptionof streamlined flow through the region without creating turbulence. Thisresults from the random arrangement of posts of different sizes and therelative spacing of the posts throughout the collection region.Relatively smooth, non-streamlined flow without dead spots is achievedat a preferred liquid flow rate of between about 0.3 to 10 mm/sec, andmore preferably the flow rate is maintained between about 0.5 and 5mm/sec and is achieved by suction from an inlet well of defined size.

The present invention also provides a method for detecting a target cellin a biological sample using the devices described herein. For example,the method may comprise contacting a biological sample with a firstbinding entity to form a pre-loading mixture, wherein the first bindingentity specifically binds to a target entity on the surface of thetarget cell, passing the pre-loading mixture through a micro-channel,wherein the surface of the micro-channel is coated with a second bindingentity capable of specifically binding to the first binding entity, anddetecting the presence of the target cell on the surface of themicro-channel. In certain embodiments, the micro-channel comprises apopulation of posts distributed on the surface of the micro-channel inrandom pattern.

Various types of biological samples, such as blood, plasma, serum, bonemarrow, semen, vaginal secretions, urine, saliva, amniotic fluid,cerebral spinal fluid, synovial fluid, lung lavages, fine needleaspirates (FNAs) and biopsy tissue samples, are suitable for use in themethods of the invention. In one embodiment, the biological sample is ablood sample from a patient. The target cell can be present in thebiological sample in the ratio of 1 out of 10¹⁰ cells, 1 out of 5×10⁷,or 1 out of 10⁴ cells. A target cell can be any cell comprising adetectable surface antigen, such as a cancer cell, stem cell, fetalcell, a viral-, a bacterial-, or a fungal-infected cell.

In one particular embodiment, the target cell is a cancer cell. Thecancer cell can be a cell from any type of cancer, such as an epithelialcancer, including, but not limited to, breast cancer cells, prostatecancer cells, colorectal cancer cells, lung cancer cells, pancreaticcancer cells, ovarian cancer cells, bladder cancer cells endometrial oruterine cancer cells, cervical cancer cells, liver cancer cells, renalor kidney cancer cells, thyroid cancer, bone cancer cells, lymphomacells (e.g. Hodgkin's lymphoma, non-Hodgkin's lymphoma), melanoma cells,and non-melanoma skin cancer cells.

The first binding entity can be any of the molecules as describedherein. In one embodiment, the first binding entity is an antibody. Thefirst binding entity may be a biotinylated-antibody and the secondbinding entity may be avidin. In some embodiments, the first bindingentity can be an antibody that specifically binds to circulatingepithelial cells. The antibody can be an epithelial cell adhesionmolecule (EpCAM) antibody, such as an antibody that specifically bindsto an epithelial cell surface adhesion protein. The first binding entitymay be a cocktail of two, three, four, five, or more antibodies, forexample, as described herein for capture of target cancer cells. Forexample, the antibody cocktail may comprise at least antibody against anepithelial cell surface antigen, and at least one antibody against anantigen that is indicative of a mesenchymal phenotype, to therebyisolate cells having a range of epithelial and/or mesenchymalcharacteristics from the sample.

For example, where the target cell is a breast cancer cell, the firstbinding entity may be an antibody that specifically binds to EpCAM(epithelial cell adhesion molecule), Her2/neu (Human Epidermal growthfactor Receptor 2), MUC-1, EGFR (epidermal growth factor receptor),TAG-12 (tumor associated glycoprotein 12), IGF1R (insulin-like growthfactor 1 receptor), TACSTD2 (tumor associated calcium signal transducer2), CD318, CD340, CD104, N-cadherin or a combination (e.g., cocktail) oftwo or more thereof.

In yet another embodiment, the target cell is a prostate cancer cell andthe first binding entity is an antibody that specifically binds toEpCAM, MUC-1, EGFR, PSMA (prostate specific membrane antigen), PSA(prostate specific antigen), TACSTD2, PSCA (prostate stem cell antigen),PCSA (prostate cell surface antigen), CD318, CD104, N-cadherin or acombination thereof. In another embodiment, the target cell is acolorectal cancer cell and the first binding entity is an antibody thatspecifically binds to EpCAM, CD66c, CD66e, CEA (carcinoembryonicantigen), TACSTD2, CK20 (cytokeratin 20), CD104, MUC-1, CD318,N-cadherin or a combination thereof.

In still another embodiment, the target cell is a lung cancer cell andthe first binding entity is an antibody that specifically binds to CK18,CK19, CEA, EGFR, TACSTD2, CD318, CD104, or EpCAM or a combinationthereof. In another embodiment, the target cell is a pancreatic cancercell and the first binding entity is an antibody that specifically bindsto MUC-1, TACSTD2, CEA, CD104, CD318, N-cadherin, EpCAM or a combinationthereof. In yet another embodiment, the target cell is an ovarian cancercell and the first binding entity is an antibody that specifically bindsto MUC-1, TACSTD2, CD318, CD104, N-cadherin, EpCAM or a combinationthereof.

In another embodiment, the target cell is an endothelial bladder cancercell and the first binding entity is an antibody that specifically bindsto CD34, CD146, CD62, CD105, CD106, VEGF receptor (vascular endothelialgrowth factor receptor), MUC-1 or a combination thereof. In anotherembodiment, the target cell is an epithelial bladder cancer cell and thefirst binding entity is an antibody that specifically binds to TACSTD2,EpCAM, CD318, EGFR, 6B5 or Folate binding receptor.

The target cell may be a cancer stem cell, and the first binding entitymay be an antibody that specifically binds to CD133, CD135, CD117, CD34or a combination thereof.

In some embodiments, the target cell is a circulating cancer cell thatexpresses mesenchymal antigens and the first binding entity is anantibody (or antibody cocktail) that specifically binds to FGFR1, FGFR4,EGFR, N-cadherin, folate binding receptor, and MSC or a combinationthereof.

In some embodiments, the target cell is a circulating cancer cell thatexpresses angiogenesis surface antigens and the first binding entityincludes an antibody that specifically binds to a VEGF receptor.

In other embodiments, the target cell is a melanoma cancer cell and thefirst binding entity is an antibody that specifically binds to one ormore of the melanocyte differentiation antigens, oncofetal antigens,tumor specific antigens, SEREX antigens or a combination thereof.Examples of melanocyte differentiation antigens, include but are notlimited to tyrosinase, gp75, gp100, Melan A/MART 1 or TRP-2. Examples ofoncofetal antigens include antigens in the MAGE family (MAGE-A1,MAGE-A4), BAGE family, GAGE family or NY-ESO1. Examples oftumor-specific antigens include CDK4 and β-catenin. Examples of SEREXantigens include D-1 and SSX-2.

In certain embodiments, the first binding entity is an antibody directedto mutated peptides that are activated as a result of cellulartransformation. These peptides include but are not limited to mutatedintrons, N-acetylglucosaminyltranferase, V gene product, MUM-1 and p15.

In other embodiments, the first binding entity is an antibody thatrecognizes the ganglioside, GM2, GD2, GM3 and/or GD3; high molecularweight chondroitin sulfate proteoglycan, CD146, or p97melanotransferrin.

In certain embodiments, the target cell is a circulating tumor cell(CTC). A CTC in the blood sample is a tumor cell is often defined bystaining positive for CK and DAPI and is staining negative for CD45(CK⁺, CD45⁻, DAPI⁺), whereas lymphocytes are CD45⁺. Detection of theCTCs in the blood circulation can aid disease management, including theability to monitor treatment efficacy or failure. However, due to thelimited number of available CTC-specific antibodies, CTCs have failed tobe captured in about 40%-60% of patient blood samples. Accordingly, thepresent invention in some aspects provides a method for capturing anddetecting these rare CTCs.

In some embodiments, the first binding entity is a mixture (e.g.,cocktail) of at least a first antibody and a second antibody, whereinthe first antibody specifically binds to a first epitope of the targetentity and the second antibody specifically binds to a second epitope ofthe target entity. The first and second epitopes can be present on thesame antigen (molecule) or the first and second epitopes can be presenton different antigens (molecules).

In one embodiment, the first binding entity can be a mixture of a firstantibody and a second antibody, wherein the first antibody specificallybinds to a stem cell antigen and the second antibody specifically bindsto a cancer cell antigen. Stem cell antigens can be present on cancerstem cells, and antibodies directed to these stem cell antigens can beadded as general capture antibodies to one or more antibodies directedto cancer antigens, such as those described herein. In some embodiments,the first antibody specifically binds to CD133, CD135, CD117, CD34 orcombinations thereof, and the second antibody specifically binds to acancer antigen.

In another embodiment, the first binding entity can be a mixture of afirst antibody and a second antibody, wherein the first antibodyspecifically binds to a mesenchymal marker and the second antibodyspecifically binds to a cancer cell antigen. Circulating tumor cells candownregulate epithelial markers and upregulate mesenchymal markers, andthus can be captured by antibodies that specifically bind to suchmesenchymal markers. In some embodiments, the first antibodyspecifically binds to FGFR1 (fibroblast growth factor receptor 1),FGFR4, MSC (mesenchymal stem cell antigen), EGFR, N-cadherin, folatebinding receptor or combinations thereof, and the second antibodyspecifically binds to a cancer antigen.

In still another embodiment, the first binding entity can be a mixtureof a first antibody and a second antibody, wherein the first antibodyspecifically binds to an angiogenesis marker and the second antibodyspecifically binds to a cancer cell antigen. In certain embodiments, thefirst antibody specifically binds to a VEGF receptor, and the secondantibody specifically binds to a cancer antigen.

In another embodiment of the invention, the method further comprisescontacting the pre-loading mixture with a third binding entity. Thefirst binding entity may be a primary antibody, the third binding entitymay be a secondary antibody conjugated to a detectable or capturableentity, and the secondary antibody specifically binds to the firstbinding entity. The second binding entity specifically binds to thethird binding entity (e.g., via the capturable entity). In anotherembodiment, the method further comprises contacting the pre-loadingmixture with a third binding entity, wherein the first binding entity isa primary antibody, the third binding entity is a biotinylated secondaryantibody that specifically binds to the first binding entity, andwherein the second binding entity is an avidin molecule. The secondaryantibody may be a whole or an intact antibody, or fragment thereof, suchas Fab′2, Fab′ or Fab, or any antibody derivatives. A derivatizedantibody can be a fragment of the antibody, an antibody that has beenconjugated to a fatty acid, carbohydrate, peptide, a chemical entitysuch as a fluorescein, streptavidin etc. A derivatized antibody can bean antibody where the amino acids have been modified to increase theavidity or affinity of the antibody to the target protein.

In some embodiments, the method further comprises cross-linking thetarget cell bound to the surface of the micro-channel. Severalcross-linking agents can be employed to cross-link the bound targetcells to the micro-channel, for example via, amino groups (amide, amineetc.), carbonyl groups, acyl groups, alkyl groups, aryl groups,sulfhydryl groups, and others that are well known to one skilled in theart. Examples of cross-linking agents include, but are not limited to,hydrophilic homobifunctional NHS crosslinking reagents (e.g.Bis(NHS)PEO-5 (bis N-succinimidyl-[pentaethylene glycol] ester) tocrosslink primary amines, homobifunctional isothiocyanate derivatives ofPEG or dextran polymers, glutaraldehyde, heterobifunctional crosslinkerscontaining NHS on one end and maleimide on the other end of the polymer;peroxide treated carbohydrate polymers to form reactive aldehydepolymers, and EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride) to crosslink carboxyl groups to primary amines. Thelength of the cross-linkers may be varied by adding one or morepolymeric units between the two reactive groups on either end of thelinker. Suitable polymeric units include, but are not limited topolymeric ethylene glycol, carbon chains, polynucleotides, polypeptides,and polysaccharides.

The cross-linking reagent can be applied to the micro-channel followingcapture of the target cells. In some embodiments, a second cross-linkingtreatment is employed following labeling (e.g. fluorescent labeling) ofthe captured cells to cross-link the label to the captured cells. Theconcentration of the cross-linking agent and duration of treatment willdepend on the type and reactivity of cross-linking reagent, type oftarget cell, binding entities employed to capture the cells, andexpression level of surface antigen to which a binding entity binds.Suitable concentrations can be from about 0.01 mM to about 10 mM, morepreferably from about 0.5 mM to about 5 mM, or most preferably about 1mM. Duration of treatment with the cross-linking reagent can be fromabout 5 min to about 120 min, about 15 min to about 90 min, or about 30min to about 60 min. Optimization of the concentration of cross-linkingreagent and duration of treatment is within the skill of the ordinaryartisan.

Detecting the presence of captured cells can be by one of severalmethods known to those skilled in the art. In one embodiment, capturedcells can be visualized by photomicroscopy. In another embodiment,captured cells may be labeled with a fluorescent molecule or stained andvisualized by fluorescent microscopy or by measuring a fluorescentsignal. For instance, captured cells may be stained with the nuclear dyeDAPI and subsequently visualized by fluorescence microscopy. In anotherembodiment, detecting the presence of the target cell is carried out bydetecting the presence of the first binding entity. Detection of thefirst binding entity can include exposing the captured cells to a taggedmolecule that recognizes and binds the first binding entity. Forexample, the tagged molecule may be an antibody labeled with afluorescent tag or colored latex particle that binds to the firstbinding entity. In one embodiment, the first binding entity is abiotinylated antibody and the tagged molecule is fluorescently labeledavidin. In some embodiments, the tagged molecule may be the same type ofmolecule as the second binding entity. In embodiments where a thirdbinding entity is present, the detection of the captured cells cancomprise detecting the presence of the third binding entity. In suchembodiments, the tagged molecule recognizes and binds to the thirdbinding entity. For example, the first binding entity can be a mouseantibody, the third binding entity can be a biotinylated secondaryantibody that binds to mouse antibodies (e.g. a goat derived anti-mouseantibody), and the tagged molecule can be either a fluorescently labeledavidin or a fluorescently labeled antibody that binds to the thirdbinding entity (e.g. a rabbit derived anti-goat antibody).

In some embodiments, subsequent analysis of the captured cells may bedesired. In one embodiment, captured cells can be released from themicro-channel and collected for further analysis. Several methods forreleasing the captured cells are known in the art and can includemechanical means (e.g. high fluid flow), chemical means (e.g. change inpH), or use of enzymatic cleavage agents. For example, a reagent may beapplied to the micro-channel to cleave the second binding entity or tocleave the bond between the second binding entity and the cells in orderto release the target cells from the micro-channel. For instance,trypsin, proteinase K, collegenase, or a specifically focused enzyme maybe used to degrade the second binding entity (e.g. antibodies,streptavidin) and/or the cell surface antigens. During such cleavage,the outlet from the micro-channel is connected to a reservoir or othercollector, and the discharge stream carrying the released cells iscollected for further analysis. Such further analysis may include, butis not limited to, detection of aneuploidy (including monosomy ortrisomy of, for example, chromosomes 1, 3, 4, 7, 8, 11, and/or 17), geneamplification, detection of gene mutation, gene duplication and othernucleic acid or protein changes well known in the art. For example, agene mutation can be a substitution, addition, deletion of one or morenucleotides in a gene sequence. In one embodiment, the nucleic acid,such as DNA or RNA, obtained from the released cells can be subjected tofluorescent in-situ hybridization (FISH), PCR analysis, RFLP(restriction fragment length polymorphism) analysis, DNA sequencing,etc. In another embodiment, proteins or glycoproteins, includingpeptides and amino acids obtained from the released cells can besubjected to, for example but not limited to, amino acid or peptideanalysis or sequencing, GC-MS and other techniques known to thoseskilled in the art of protein analyses. In yet another embodiment, thecaptured cell released from the micro-channel device can be analyzedmorphologically by light microscopy, electron microscopy, scanningmicroscopy, immunocytochemistry staining (ICC) for internal cellularstructures or surface proteins expression, etc.

In another embodiment, the captured cells may be further analyzed insitu. For example, the cells may be counted while attached, labeled withfluorescent markers, subject to in situ hybridization analysis, such asFISH. Because antibody-antigen bonds are not covalent, they can bedissociated under some circumstances. Therefore, in some embodiments, itis highly desirable to further stabilize the cells on the micro-channelby crosslinking the cells to the channel so that cells are not dislodgedand lost during the various in situ labeling, heating, denaturing andwashing steps. Cross-linking can be a particularly importantconsideration with cells that express a low level of the surfaceantigens targeted by the first binding entity since these cells can bemore weakly attached to the second binding entity. Covalent crosslinkingof the cells to the channel surface matrix can stabilize captured cellsduring post-capture analysis.

In another aspect, the invention provides a method of post-captureanalysis of circulating cells. The circulating cells may be captured asdescribed herein, including by the methods or devices of the invention.In some embodiments, the circulating cells are captured and evaluatedwithout one or more enrichment and/or cell replicating or duplicatingprocesses, e.g., via cell culture, etc. In this aspect, circulatingcells are evaluated for malignancy independent of CK status orexpression, e.g., without CK staining and/or any other evaluating assay.For example, in accordance with this aspect, captured cells areevaluated (as described herein) for aneuploidy. The aneuploidy may bewith respect to, for example, chromosomes 1, 3, 4, 7, 8, 11, and/or 17.In certain embodiments, the invention involves evaluating circulatingcells for monosomy or trisomy 8, 11, and/or 17. In certain embodiments,the invention involves evaluating circulating cells for monosomy 8, 11,and/or 17. Aneuploidy may be detected using any know method, such asFISH. Additionally markers of cancer or malignancy may be used (exceptcytokeratin expression), such as those described herein, including Her2expression.

This invention is further illustrated by the following additionalexamples that should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety.

EXAMPLES Example 1 Construction of Basic Micro-Channel Device

One embodiment of a micro-channel device for separating biomolecules orcells is shown in FIG. 1. The device comprises a substrate or support 11which is formed with a flow path that includes a micro-channel 13 towhich sample liquid is to be supplied through an opening or well 15 thatserves as an entrance or inlet at a first end of the device and anopening 19 that serves as an outlet at the second end of the device. Thecross-section of the collection region 17 is greater than that of aninlet section 18 that leads there into from the inlet opening 15. Theinlet section contains one or more pairs of axially aligneddivider/supports 21 just upstream of where it widens at the end of theregion 18 to enter the collection region 17. These central dividersbreak the flow into two or more paths and serve to distribute the flowof liquid more evenly as it is delivered to the entrance end of thecollection region 17. The collection region contains a plurality ofupstanding posts 23 that are aligned transverse to the liquid flow pathand arranged in an irregular, generally random pattern across the entirewidth of the collection region portion of the flow channel. The patternof the posts is such that there can be no straight-line flow through thecollection region and that streamlined flow streams are disrupted,assuring there is good contact between the liquid being caused to flowalong the flow path and the surfaces of the posts. The posts areintegral with the flat base of the collection region 17 and extendperpendicular from the base, presenting surfaces that are verticalrelative to a horizontal path of the liquid being caused to flow throughthe flow channel of the substrate 11. Another flow divider/support 21 ais located at the exit from the collection region.

The substrate is formed from PDMS and is bonded to a flat glass plate toclose the flow channel. The interior surfaces throughout the collectionregion are derivatized with amine groups (Inventors: Is the amine groupspecific to PDMS or can there be other active groups, e.g. SH— that canbe derivatized. I seem to remember one can coat supports with polylysineto attach cells or positively charged proteins) by incubating for 30minutes at room temperature with a 3% solution of3-aminopropyltriethoxysilane. After washing with ethanol the aminegroups on the channel are derivatized for 30 minutes with bifunctionalPEG linker molecule containing an NHS ester on one end and a maleimidegroup on the other end. In this reaction the NHS group reacts with theamine groups on the channel. After washing the channel with PBS asolution of 0.5 mg/mL thiolated streptavidin is added which will reactwith the maleimide groups on the other end of PEG linkers attached tothe channel. Thiolated streptavidin is prepared by treatment ofstreptavidin with Traut's reagent as is commonly known in the art. Afterincubation for 60 min, the excess thiolated streptavidin is washed fromthe micro-channel with PBS/1% BSA and stored for future use.

In a typical example, 10 mLs of blood is obtained and the buffy coat isisolated by density gradient sedimentation as is commonly known in theart. The buffy coat contains the nucleated white blood cell fraction ofthe blood and also contains epithelial or other nucleated cells presentin the blood. The buffy coat contained in a volume of approximately 0.5mL in a centrifuge tube is incubated with the first binding entity ofthe present invention for 30 min, and then the tube is filled withapproximately 30-fold excess of PBS/BSA and centrifuged to pellet thebuffy coat cells. The sample is resuspended in approximately 200 μL andpassed through the avidin-coated micro-channel by hooking themicro-channel device up to outlet tubing from a syringe pump which isfilled with about 50 μL of the cell suspension. The syringe pump isoperated to produce a slow continuous flow of the sample liquid throughthe micro-channel device at room temperature and a rate of about 10μL/min. During this period, the avidin attached to the surfaces in thecollection region where the random pattern of transverse posts arelocated, captures the target cells of interest in the sample. After theentire sample has been delivered by the syringe pump, a slow flushing iscarried out with a PBS/1% BSA aqueous buffer. About 100 μL of thisaqueous buffer is fed through the device over a period of about 10minutes, which effectively removes all non-specifically boundbiomaterial from the flow channel in the device. Two additional washingsare then carried out, each with about 100 μL of PBS/1% BSA over a periodof about 10 minutes.

At this time, inasmuch as the device is made of optically clearmaterial, microscopic examination can be made of the effects of thecapture using photomicroscopy. Captured cells may be treated furtherwith additional antibodies and fluorescent probes and analyzed byfluorescence microscopy.

Example 2 Comparison of Cell Capture Rates Between Pre-LabeledMicro-Channels and Pre-Labeled Cells

As described in U.S. Published Application No. 2006/0160243, filed Jan.18, 2005 and elsewhere (Nagrath et al. (2007) Nature, Vol.450(7173):1235-9), previous devices for capturing cells of interestcomprised a micro-channel that was derivatized with an antibody that wasspecific to antigens on the cells of interest. The suspension containingthe rare cells of interest was then passed through the channel and cellswere captured by the cell-specific antibody (FIG. 2).

While the level of antigen expression can be determined in culturedcells and on clinical tissue samples such as tumors, it is not knownprecisely how many antigens are available on the surface of acirculating tumor cell (CTC). It is known that tumors are highlyheterogeneous and that cells detached from the tumor into the blood canchange their expression levels of antigen. Therefore, it is most likelythat CTCs are a highly heterogeneous population with specific antigenlevels varying from very low to very high in any given sample. To obtainmaximum capture of CTCs from a sample, it is best to optimize the systemto capture cells with the lowest antigen expression levels.

The devices of the present invention comprise a micro-channelderivatized with a general antibody or protein that can bind tocell-specific antibodies as described in Example 1. The cell-specificantibody is added to the sample containing the cells of interest priorto passing the sample through the micro-channel, thus pre-labeling thecells. The cells of interest are then captured when the general antibodyor other protein coating the channel binds to the cell-specific antibodybound to the cells of interest (FIG. 3).

The following set of experiments were conducted to determine whetherpre-labeling a sample containing CTCs with antigen-specific antibodiesresult in a better capture rate on micro-channel devices as compared tomicro-channel devices coated with the antigen-specific antibody. Acommon antigen used to capture CTCs is EpCAM, an epithelial cell surfaceadhesion molecule. For these experiments, the bladder cell line, T24,was used, which is known to express low levels of EpCAM.

In the traditional device, the micro-channel was derivatized withstreptavidin and then biotinylated antibody for EpCAM was pre-loadedonto the channel (EpCAM channel). The EpCAM antibody was able to bindthe EpCAM antigen on the surface of the T24 cells, thus capturing thecells in the micro-channel. In the device of the present invention, themicro-channel was derivatized with streptavidin (Strep channel) and thebiotinylated antibody for EpCAM was incubated with the sample of T24cells at approximately 1 μg/mL for 30-60 mins. prior to passage of thecells over the streptavidin-coated channel. The streptavidin binds tothe biotinylated EpCAM antibody bound to the surface of T24 cells, thuscapturing the cells in the micro-channel. Thus the reagent components ofthe two devices are identical except that they are applied to thedevices in a different order.

As shown in FIG. 4, use of the streptavidin-coated channel with cellspre-incubated with the biotinylated EpCAM antibody unexpectedly producedcapture percentages about twice as high as those obtained with the EpCAMchannel and unlabeled cells. The increased capture percentage is about2-3 fold higher when cells are passed through the channel under multipleflow rates.

In a next series of experiments, 1.2 μg/mL of the biotinylated-EpCAMantibody was pre-incubated with the cells for 30 mins. Thisconcentration of antibody was about 100 to 1000 fold molar in excessover the total antigens present on the T24 cells and therefore,significant excess antibody remained in each suspension. After a 30mins. incubation, the excess antibody was diluted to less than 0.05μg/mL by dilution of the cells to approximately 200 cells forapplication on the channel. This sample of cells served as a controlsample and was applied directly to the channel in 250 μL PBS/BSA (sampleA in FIG. 5). In samples B-D excess antibody at the indicatedconcentrations were added back to the 250 μL cell suspension prior torunning on the channel. As shown in FIG. 5, free antibody does notinterfere with binding to the streptavidin on the channel, and does notdecrease cell capture as expected, but in fact increases cell capture.

In a similar experiment, the volume of the initial cell suspensionapplied to the channel was increased from 250 μl to 2 mL. Since the μgper mL of the added extra antibody remained the same as in FIG. 5, thetotal μg of absolute antibody in the sample with approximately 200 cellswas nearly 10 times higher than in FIG. 5. As shown in FIG. 6, the addedextra antibody shows a similar increase in cell recovery relative to theconcentration of the antibody that was observed in the results depictedin FIG. 5. This result indicates that the observation of higher recoveryis related to the concentration of excess antibody in the cellsuspension and not the absolute μg of total antibody in the cellsuspension.

The results of this series of experiments show unexpected advantages incollecting cells of interest in a micro-channel flow device when thecells are pre-labeled with antibody. As seen in FIG. 4, pre-incubatingthe cells with antigen-specific antibody significantly improves capturein the micro-channel device as compared to capture in micro-channelscoated with the antigen-specific antibody. In addition, the presence ofexcess antibody in the cell sample during the run does not limit thismethodology but can in fact mediate increased binding of the cellularantigens to the streptavidin matrix on the channel, thereby enhancingcapture.

Example 3 Use of Multiple Antibodies Increases the Capture Rate ofTarget Cells

It has traditionally been considered most efficient to pre-load anantibody onto the channel. However, the negative effects on cell captureof loading a channel with multiple antibodies have not been previouslyconsidered. An advantage of using a micro-channel coated with a generalbinding partner (e.g. antibody or protein) of an antigen-specificantibody is that multiple antibodies can be added to a cell suspensionto pre-label cells without lessening the availability of any singleantibody. Because multiple antigen sites on a cell are not mutuallyexclusive, when adding multiple antibodies to the cell suspension thecapture efficiency on the channel is not diminished for any singleantibody. By way of example, if the channel could accommodate 100antibody sites and a mixture of 5 different antibodies were added tocoat the channel, then each antibody would occupy ˜20% of the channelspace. Thus, the potential binding efficiency for each individualantibody is only 20% of what it would be if it covered the entirechannel. Regardless of the number of antigens on the cell, the channelis inherently less efficient at capturing those cells with only 20% ofthat individual antibody. When the cell has a low number of targetantigens, the efficacy in capturing these low antigen expressing cellscan be amplified by the addition of the antibodies specific for othertarget antigen in the cell suspension prior to binding to the substrateor support of the micro-channel device. For example, if the same 5antibodies are added to the cell suspension, then each antibody canmaximally bind to all cognate cell surface antigens independently,without interference or reduction due to the presence of otherantibodies bound to different epitopes on the cell. By derivatizing eachof the five different antibodies with a common capture tag (e.g.biotin), a channel coated with a binding partner for the capture tag(e.g. streptavidin) can bind all 5 antibodies simultaneously to theirrespective antigens on the cell, thus producing an additive effect oncell capture.

FIG. 7 shows a reduction in the capture of T24 cells when the ratio ofEpCAM antibody to murine IgG on the channel is lowered when theantibodies are first coated on the substrate/support of themicro-channel device. To determine the effect of EpCAM capture in thepresence of additional biotinylated antibodies, the biotinylated EpCAMantibody was diluted with irrelevant biotinylated mouse IgG and theresulting mixture was used, either to coat the channel with antibody oradded to the cell suspension prior to passage over the channel. FIG. 7(sample A) shows that the percentage of T24 cell-capture is about twiceas high when the cells were pre-labeled with biotinylated EpCAM antibodyonly. This observation is consistent with the results seen in FIG. 4.However, when the EpCAM antibody was diluted in a 1:1 ratio with anirrelevant antibody and used to either label the cells directly or tocoat the channel, the channel recovery drops from 24% to 7%, while therecovery of pre-labeled cells is unaffected (FIG. 7, sample B). When theEpCAM was diluted in a 1:4, the recovery drops to 1% when the antibodymixture was first coated on the channel while the recovery is unchangedwhen the cells were pre-labeled with the antibody mixture prior tobinding to the substrate or support of the microchannel device (FIG. 7,sample C). These results demonstrate that dilution of the EpCAMantibodies by additional antibodies do not interfere with the maximalbinding of the EpCAM antibodies to the cells when the cells wereprelabeled with the soluble antibodies, but that precoating of thechannel with the diluted EpCAM antibodies shows a significant reductionin the capture of the low EpCAM expressing T24 cells. It is thereforeevident that if the EpCAM antibody were mixed with 2 or 3 or 4 differentantibodies for binding on the channel, even if the other antibodies wererelevant to a surface antigen on the cell, the EpCAM antibody itselfwould be commensurately diminished in its binding effectiveness.Therefore, when adding multiple antibodies to the channel, the effect ofeach antibody cannot be expected to be additive. The overall effect oncell capture is unpredictable in this configuration since circulatingtumor cell (CTC) antigen levels are variable. By definition the antibodyin a mixture that might be directed towards the highest level antigen onthe CTC will be diminished by the addition of antibodies to the lesserantigen levels on the CTC. If only one antibody in a mixture recognizesa dominant epitope on a particular CTC, then diluting with several otherantibodies on the channel will adversely affect capture instead ofenhancing it. By contrast, mixtures of soluble antibodies added to cellsprior to passage over the channel are additive.

To demonstrate the additive effect of multiple antibodies on prelabeledcells prior to passage over the channel, two different antibodies to twodifferent cell surface adhesion antigens, Trop-1 and Trop-2, were addedto cell suspensions of either T24 bladder cells or SKOV ovarian cells.Each of the antibodies was biotinylated and cells were captured using amicro-channel device coated with streptavidin. When Trop-1 antibody wasused to pre-load T24 cells, 29% of the cells are captured (FIG. 8). WhenTrop-2 antibody, which binds to a different antigen than Trop-1antibody, was added in combination with the Trop-1 antibody, 94% of thecells are captured. A similar result is obtained with SKOV cells. Acapture of 74% of the cells is observed with pre-labeling with Trop-1antibody alone. However, a capture of 89% of the cells is observed whenboth Trop-1 and Trop-2 antibodies were added simultaneously (FIG. 8).The results show that addition of more than one antibody to more thanone target site on the surface of the cell increases the effectivenumber of channel-detectable molecules attached to the target cell andproduces an additive effect on cell capture.

In FIG. 9 the same additive effect is observed using a different cellline and with a different antibody mixture. In this case the breastcancer cell line, MDA-MB-231, which has a low EpCAM expression wastested. In FIG. 9, the % capture with EpCAM antibody alone is low, butadding a mixture of 6 antibodies specific for the antigens: EpCAM,Trop-2, EGFR, MUC-1, CD318 and HER-2 improves capture to essentially100%. FACS analysis of the MDA-MB-231 showed that this cell line hasvery low antigen expression of EpCAM, Trop-2, Her-2, and MUC-1 buthigher expression of EGFR and CD318. Therefore, the antibodies to thehigher expressing antigens were diluted 3-fold with antibodies to lowexpressing antigen. The diluted antibodies are still highly effective incapturing this low EpCAM-expressing cell line. This result is consistentwith the results shown in FIG. 7 where antibodies were used to pre-labelthe cells.

Example 4 Secondary Antibody Labeling of Target Cells Can Effect Capturein the Micro-Channel Device

In some instances, a non-derivatized primary antibody may moreefficiently bind to the antigen of interest or may be easier to employ.With some antibodies their activities are adversely affected byderivatization procedures which modify their surface amino acids. Incases, where one desires to use a non-derivatized primary antibody tobind to cellular antigens, a derivatized secondary antibody may be addedto the cell suspension to form a complex with the primary antibody whichis bound to the cellular target antigen. Thus, primary antibodymixtures, semi-purified or non-clonal hybridoma supernates can be addedto the cell suspension and any antibodies that attach to antigens on thecell can be labeled by the addition of a derivatized (e.g. biotinylated)secondary antibody. Antibodies that do not bind to the cell are simplywashed away.

To illustrate this approach, the cultured ovarian SKOV cell line waspre-labeled with either biotinylated Trop-1 antibody (Sample A in FIG.10) or non-biotinylated Trop-1 plus a 3-fold molar excess ofbiotinylated anti-mouse secondary antibody. The primary antibody(Trop-1) concentration was 1 μg/mL and the cells were incubated for 30mins. either with or without 3 μg/mL secondary antibody before cellcapture and purification on a micro-channel device. The differencebetween samples B and D was that a longer biotin linker on the secondaryantibody was used in sample D. In sample C, the cells were washed withPBS/BSA to remove excess primary and secondary antibody before applyingthe cells to the channel. In all samples, approximately 200 cells weresuspended in 250 μL of PBS/BSA for application to the channel. As shownin FIG. 10, all samples have similar recovery. These results demonstratethat biotinylated secondary antibody can be used in combination withunlabeled primary antibody to pre-labeled cells for effective capture ina micro-channel device. The presence of some excess biotinylatedsecondary antibody did not adversely affect the capture percentagecompared to direct pre-labeling with 1 μg of biotinylated Trop-1.Secondary antibodies may include intact IgG antibody, or antibodyfragments such as Fab′2, Fab′, Fab or engineered antibody fragments suchas single chain Fab or single chain variable fragment.

Example 5 Stabilization of Captured Cells on Channel Surface

The process of capturing cells on a micro-channel device involves flowof cells suspended in a liquid. Therefore, the cells are subjected tosheer forces from the liquid flow that can also dislodge the cells fromthe channel after they are captured. This effect is more pronounced withcells that have lower surface antigen levels because there arerelatively fewer attachment points between the cell and the specificcell surface antigens bound to the channel surface by the antibody.Therefore, it is advantageous to provide an additional externalattachment of the cell to the channel surface by means of cross-linkingreagents to better stabilize the attachment of the cell to the channel.Since the channel is typically coated with a binding protein (e.g.streptavidin or an antibody), a facile means of further anchoring thecell to the channel is through protein cross-linking reagents.

Reagents known in the art for this purpose can be homo-bifunctional NHSesters to crosslink amino groups on proteins. Another way ofcross-linking is through the thiol or disulfide groups on the proteinswith thiol reactive reagents, such as heterobifunctional molecules witha maleimide and an NHS ester. In addition, reagents such as EDC can beused to cross-link carboxyl and amino groups. The length of thesecross-linkers can be varied by the use of polymeric regions between thetwo reactive groups, which typically take the form of chemical linkerssuch as polymeric ethylene glycol or simple carbon chains, but can alsoinclude sugars, amino acids or peptides, or oligonucleotides. Polymerchain lengths of from 5 to 50 nm are typical for this purpose but can beshorter or longer as needed. The common property of all of these proteincross-linking reagents is to covalently bind cellular proteins so as toanchor the cell to the surface of the channel by multiple covalentattachment points.

To examine whether externally added cross-linking reagents enhanceretention of the captured cells on the coated micro-channels, cells werecaptured on coated micro-channels and subjected to high flow rates inthe absence or presence of a protein cross-linker. Streptavidin-coatedsurfaces of micro-channels were prepared. The cultured T24 cell line,which is known to have a low expression level of surface EpCAM, was usedas a model cell line. One μg/mL biotinylated anti-EpCAM antibody wasincubated with the cells for 30 mins. at 4° C. and approximately 325cells were suspended in 250 μL of PBS/BSA buffer and passed intriplicates over coated micro-channels at 12 μL/min. The exact number ofcells applied to the channel was determined microscopically by countingthe cells in duplicate aliquots. After the cell suspension was passedthrough the channel, the channel containing bound cells was washed oncewith PBS/BSA and then a solution of homobifunctional NHS ester (bisN-succinimidyl-[pentaethylene glycol] ester) at 2 mM was passed over thechannel and allowed to incubate for 20 mins. The control channel withoutNHS ester received only PBS/BSA solution. The cells were then washedwith a 5% PEG solution in PBS for 2 mins. at various flow rates. The 5%PEG/PBS solution increases the solution viscosity and along with higherflow, provides more sheer force on the cells for purposes of thiscomparison. The cells captured in the channel were then stained with thenuclear staining dye, DAPI and counted.

FIG. 11 shows photomicrographs of captured cells subjected to differentflow rates in the absence of protein cross-linker. Almost 50% of thecells are lost at a flow rate of 20 μL/min and all of the cells are lostat a flow rate of 100 μL/min.

FIG. 12 shows photomicrographs of captured cells subjected to differentflow rates after exposure to a NHS protein cross-linker. All cells areretained on the channel at flow rates of up to 50 μL/min and only onecell was lost at a flow rate of 100 μL/min.

A quantitative comparison of capture with and without cellularstabilization by protein cross-linking is shown in FIG. 13. As inprevious experiments, approximately 200 T24 cells were applied to themicro-channel and after capture, the cells were washed with 5% PEG inPBS. FIG. 13 shows that less than 50% of the cells which not treatedwith crosslinking reagent are recovered compared to the percentage ofcells recovered in channels treated with a crosslinker. Thus, theaddition of protein cross-linking reagents significantly stabilizes cellattachment to the micro-channel. It should be noted that this result isindependent of how the cells were captured on the micro-channel, whetherby pre-loading antibody on the cells or on the channel, since thecrosslinking agent stabilizes the cell on the micro-channel after thecells have been captured.

A second experiment similar to the above was employed to test for cellstability on the channel. After treatment of the cells with the proteincrosslinker as above, the SKOV cells on the channel were subsequentlystained with anti-cytokeratin (to visualize epithelial cells) and DAPI(to visualize cells with a nucleus). The difference in this experimentwas that the tubing connected to the outlet was disconnected, a processthat can cause transient but abrupt pressure pulses that can sheer anddislodge cells from the micro channel.

Table 1 shows the increasing numbers of cells lost when cells were notcrosslinked to the channel were subjected to exogenous mechanical forcesas a result of removing the outlet tubing. If cells were fixed to thechannel with methanol treatment prior to removal of the tubingconnections, there is no significant difference in cell recoveryregardless of whether crosslinker was used (data not shown). However,methanol fixation (or any alcohol or acetone fixation) has severalundesirable side-effects for the purposes of some subsequent cellanalyses. Cells fixed with methanol are permeabilized due to disruptionof the cell membrane and therefore cell surface studies cannot bedistinguished from internal cell reactions. In addition, cells fixedwith methanol become fused to the channel matrix making cell removaldifficult and inefficient. Such cells can be subjected to extensiveproteolysis to aid in cell removal, but cellular digestion has severalundesirable side-effects for some types of subsequent cellular analysis.The procedure of crosslinking cells to the channel allows stabilizedcells on the channel to be retained without alcohol fixation duringnormal channel operations and manipulations including higher flow rates,higher viscosity buffers and removal of channel connections.

TABLE 1 2 mM 2 mM 0.2 mM 0.07 mM Crosslinker + Crosslinker CrosslinkerCrosslinker Conditions methanol No methanol No methanol No methanol %retained 100% (control) 96% 60% 28% on channel

Example 6 Antibody Mixtures (Antibody Cocktail) Enhances Capture ofEpithelial-Like and Mesenchymal-Like Cancer Cells

Urothelial carcinoma (UC) cell lines have lower expression of EpCAM inmore invasive tumor models. Such cells in circulation would be expectedto limit the utility of EpCAM-based CTC capture. A cohort of 5 UC celllines (UMUC3, UMUC5, UMUC9, T24, and KU7) were selected based on geneexpression heat map analysis as being either more epithelial or moremesenchymal-like. In the latter case, these cells have undergone theepithelial to mesenchymal transition (EMT) which results in epithelialcells with mesenchymal expression and morphological characteristics.This EMT has been proposed as a mechanism by which epithelial cells candissociate from the tumor and become more migratory and invasive incirculation.

These EMT cells were further tested by FACS for a variety of cellsurface antigens. After identifying expression differences in these celllines, an antibody mixture of EpCAM and 5 additional antibodies wasselected to improve cell capture of all UC cell types. We subsequentlycompared cell capture rates using microfluidic channels with theantibody mixture compared to EpCAM alone. Cells were also immunostainedwith cytokeratin and vimentin antibodies to help further distinguishcells having epithelial or mesenchymal-like expression characteristics,respectively.

FIG. 14 shows the staining of the 5 UC cells lines with vimentin andcytokeratin. Among the 5 UC cell lines, 2 (UC3 and KU7) stained withvimentin and had minimal to no expression of EpCAM. While these cellslines retained some degree of cytokeratin staining, one cell linestained only with vimentin (FIG. 14). The remaining 3 lines (UCS, UC9and T24) stained only for cytokeratin and had significant EpCAMexpression. Those cell lines with no EpCAM expression (UC3 and KU7) hadno cell recovery when EpCAM alone was utilized as the capture antibody.However, when the antibody mixture, comprising 6b5, CD318, EGFR, MOV18,Trop-2 and EpCam) was used, all 5-cell lines achieved nearly 100% cellcapture rates. In the case of KU7, the most mesenchymal-like of thisgroup of cell types, the folate binding receptor (MOV18) was unique andnot expressed in the other cell lines.

The results show that the use of a mixture of antibodies allows captureof both bladder epithelial cells and bladder epithelial cells that hadundergone EMT. The study shows that the use of antibody mixturesprovides a dramatic improvement over cell recovery compared to the useof a single antibody alone, such as EpCAM alone. Because of theheterogeneity of tumor cell types expected in circulation, such anapproach is expected to significantly improve the capture and isolationof CTCs from patient samples.

Example 7 Capture of Low-Antigen Expressing Cells on a Micro-ChannelDevice Increases with Antibody Mixture or Cocktails

Common detection methods are needed when cocktails of antibodies areused to simultaneously bind to several different cancer cell types.While cytokeratin stain works well for epithelial cells, some epithelialcells have lost cytokeratin expression as described in Example 6. Withother cells types, such as stem cells, there is no specific method forstaining these cells that does not have significant crossreactivity toother blood cell types which may be non-specifically bound to thechannel. However, high levels of biotinylated primary or secondaryantibodies on the surface of the cells are common to all cells capturedspecifically by the avidin on the microchannel. The benefit of usingcocktails of biotin-conjugated antibodies is the additive effect inincreasing surface biotins on target cells, which is useful forincreasing the capture of low antigen-expressing cells or cellsexpressing variable levels of one or more antigens in a heterogenouscell population, such as those found in tumor patients. See FIGS. 8, 9,and 13.

The unexpected additional advantage of using multiple antibodies in acocktail is that this provides a common detection method for aheterogenous population of cells that have variable level of antigenexpression. An example of this is shown in FIGS. 15A-15B.

FIG. 15A shows the percentage capture of SKOV cells, which is known toexpress a high level of EpCam antigen (approximately 40-70,000 EpCamantigens per cell (apc)), with EpCam alone or with a mixture ofantibodies specific for other surface antigens expressed by the cells,including EpCAM, Trop-2, EGFR, MUC-1, CD318 and HER-2. The results showthat there is no significant improvement in the percent number of SKOVcells captured with EpCam antibody alone or a mixture of antibodiesspecific for other antigens in addition to EpCam.

In contrast, FIG. 15B shows the fluorescence staining intensity of thesame SKOV cells by FACS and on slides. These cells stain with verydifferent intensities depending on whether they have been pre-mixed withEpCam alone (˜66,000 surface antigens) or with an antibody mixture whichare directed against Her-2, CD24, CD44, combined surface antigen levelof ˜600,000 antigens as determined by the FACS analysis. Fluorescentlylabeled anti-mouse antibody was used to label the primary antibodies.While there was minimal increase in the capture of these antibodycocktail-incubated SKOV cells using biotinylated secondary antibody asshown in FIG. 15A, using fluorescently labeled secondary antibody inFIG. 15B shows that the staining intensity is significantly higher whenusing the antibody mixture. In a similar manner, this differential wouldbe obtained if cells were reacted with primary antibody, followed bybiotinylated secondary antibody and fluorescently labeledbiotin-reactive avidin. Thus there is a significant advantage in usingantibody cocktail mixtures even when additional antibodies are notnecessary for capture of the cells. In the case of a lowEpCam-expressing cells, the capture using EpCam antibody alone isreduced (FIGS. 4-6, 14), but is significantly increased when using anantibody cocktail. In this case the staining intensity based on thenumber of antibodies bound to the surface of the cells would also beincreased. Therefore the use of fluorescently-labeled molecules thattarget the multiple antibodies used to better capture the cells has theuniversal advantage of better detection of those same cells. The use ofantibody cocktails has the unique advantage in allowing detection ofcells for which there may not be a known specific marker for detectionsuch as cytokeratin in epithelial cells, or where the cytokeratin hasbeen lost as shown in FIG. 14. The multiple antibodies used in a mixturefor better capture of cells with variable expression of surface markerscan still be targeted for fluorescence labeling based solely on theirincreased levels of bound antibodies from the antibody cocktail.

FIG. 16 shows the additive effect of multiple antibodies in a cocktail,which contain antibodies specific for the SKOV target cells and whichare shown to associate minimally to the non-specific cells present in ablood sample, when the blood sample was spiked with SKOV target cells.The antibody cocktail contained antibodies directed against CD340, EGFR,CD318, Muc-1, Trop-2, EpCam, Mov-18, MSC, c-met and N-Cadherin. Althoughsome of the non-specific cells in the sample may have adsorbed some ofthe biotinylated antibodies (either primary or secondary) added to thesamples, the level of antibodies adsorbed is far too low to bevisualized using fluorescently-labeled neutravidin. The differentialstaining between specific target cells and non-specific cells favors thevisualization of the target cells which have higher numbers ofbiotinylated antibodies from the antibody mixture bound or captured bythe target cells. FIGS. 15A-15B and 16 demonstrate that addition ofmultiple antibodies in a cocktail provides a common and universal methodof detecting rare cell types that express low levels of antigens on themicrochannel. Thus, the antibody cocktail used to enhance and therebyincrease capture of circulating tumor cells that are highly variable inheterogenous cell population in a sample, also enhance detection of anyof the captured cells.

Example 8 The Micro-Channel Device is Superior at Capturing Cells fromBiological Samples that are Present in Low Cell NUMBERS

In FIG. 17, blood samples were spiked with a variable number of SKBr3cells, a cell line expressing high levels of EpCAM, ranging from about10-250 cells per 10 mL blood sample. EpCAM antibodies were added to thespiked blood sample and the EpCAM Ab-bound cells were captured on amicro-channel device using the method described in Example 1.

The results in FIG. 17 shows that approximately a 100% of the SKBr3cells were recovered from the spiked samples. The data shows that thepercent capture of cells by the micro-channel device is independent ofthe cell input.

Example 9 Antibody Cocktail is Superior at Capturing Circulating TumorCell (CTC) from Patient Blood Samples Using Micro-Channel Device

Table 2 shows the results of circulating tumor cells (CTCs) captured ona micro-channel device from 10 mL blood samples from patients diagnosedwith prostate, lung, pancreatic, renal cell, colorectal, breast andovarian cancers. The blood samples were pre-labeled with a cocktail ofsoluble antibodies containing antibodies directed against CD340, EGFR,CD318, Muc-1, Trop-2, EpCam, Mov-18, and MSC, or a anti-EpCAM only.Cells were identified by staining with fluorescently labeledanti-cytokeratin.

TABLE 2 Sample No.* Anti-EpCAM only MAb Cocktail 1 (16283 - Prostate) 02 2 (16302 - Prostate) 0 3 3 (16318 - Prostate) 95 77 4 (16291 -Ovarian) 1 1 5 (16278 - Colo/rectal) 0 4 6 (16288 - Lung) 0 5 7 (16297 -Breast) 1 3 8 (16296 - Breast) 0 3 *Samples from prostate, lung,pancreatic, renal cell, colorectal, breast and ovarian cancers.

TABLE 2 shows that the blood samples pre-labeled with a soluble antibodycocktail is superior at capturing CTCs compared to samples pre-labeledwith a single type of antibody alone.

Example 10 The Micro-Channel Device is Superior at Capturing CTCs whenBlood Samples Obtained from Breast Cancer Patients that are Pre-Labeledwith a Soluble Antibody Cocktail on a micro-Channel Device as comparedto Capture of CTCs Using a FERRO-MAGNETIC Label Antibody

Blood samples were pre-incubated with anti-EpCAM antibody for capture ona micro-channel device or pre-incubated with antibodies that are joinedto microscopic iron particles (immunoferromagnetic Abs) and capturedusing CELLSEARCH® (VERIDEX, LLC). The captured cells were stained forCK, CD45 markers and DAPI, a nuclei stain. The cells that were stainedin-situ with CK⁺/CD45⁻/DAPI⁺ were counted.

TABLE 3 Total #CTCs by CEE Sample ID (CK⁺/CD45⁻/DAPI⁺)* Veridex 16163 00 16170 0 0 16171 60 (34) 54 16172 5 0 16173 0 1 16176 549 (325) 126716187 104 (37) 54 16196 0 0 16198 87 (27) 32 16202 5 8 16203 2008 92316205 78 51 *No Significant difference by Two Tailed t-test (P = 0.715)

Total CTC counts indicated in bold include robust, apoptotic andmicronuclei; whereas numbers in parenthesis indicate robust CTCs.

TABLE 3 shows that the total number of CTCs captured on themicro-channel device that are CK⁺/CD45⁻/DAPI⁺ are consistently more thanthe CTCs captured by the VERIDEX systems, indicating that the inventionprovides for superior capturing of CTCs.

Example 11 Post-Capture Molecular Analysis of Captured Cells IncreasesIdentification of CTC as Cancer Cells in Stage III and IV Breast CancerPatients

Circulating tumor cells (CTCs) were captured from blood samples of StageIV (TABLE 4) and III (TABLE 5) breast cancer patients. The CTCs werepre-labeled with an antibody cocktail, containing antibodies to CD340,EGFR, CD318, Muc-1, Trop-2, EpCam, Mov-18, and MSC, and were releasedfrom the micro-channel device. The captured cells were analyzed byfluorescent in-situ hybridization (FISH) for aneuploidy in chromosome 8and 17, and amplification of the breast cancer marker, Her2 (TABLE 4)These cells were never released from the microchannel and all FISH isperformed in the channel with cells relocated following enumeration forFISH analysis. The total number of CTCs found positive for aneuploidywere compared to the total number of cells stained positive for CKmarker.

TABLE 4 #CTCs #Aneuploid cells Her2/chromosome Sample # (CK⁺) (Chrom 17& 8) 17 Ratio 1 3 7 (6 CK⁻) 1.05 2 1 1 (CK⁻) 1.0 3 0 3 (CK⁻) 1.0 4 0 3(CK⁻) 1.0 5 0 4 (CK⁻) 1.0 6 2 13 (CK⁻/CK⁺) Mixed 7 1 1 (CK⁻) 1.0 8 1 7(CK⁻) 0.95 9 510 7 (CK⁺) 1.0 10 16 16 (CK⁻/CK⁺) >6 11 1 2 (CK⁻) 1.0 12 04 (CK⁻) 1 13 0 2 (CK⁻) 1 14 0 14 (CK⁻) 1 15 0 1 (CK⁻) 1.5 16 0 24 (CK⁻)1.98 17 3 9 (CK⁻) 5.714

TABLE 4 shows that post-capture molecular analyses of CTCs from stage IVbreast cancer patients for aneuploidy and Her2 amplification status aresuperior in detecting breast cancer cells from the captured CTCscompared to CK staining.

In TABLE 5, captured CTCs from the blood samples of patients diagnosedwith Stage III cancer were analyzed for aneuploidy in chromosome 8, 11and 17. The total number of CTCs found positive for aneuploidy werecompared to the total number of cells stained positive for CK marker.The details of aneuploidy on chromosomes 8, 11 and 17 are shown.

TABLE 5 Sample #CTCS #Aneuploid Aneuploid Details (Chromosomes ID (CK⁺)cells 8, 11 and 17) 16610 0 93 4-Monosomy 8; 6-Monosomy 11; 83-Monosomy17 16620 0 55 26-Monosomy 8; 11-Monosomy 11; 16-Monosomy 17; 2-complexaneuploidy 16621 0 54 8-Monosomy 8; 22-Monosomy 11; 23-Monosomy 17;1-Trisomy 17 16631 0 169 11-Monosomy 8; 11-Monosomy 11; 265-Monosomy 17;3-complex monosomies 16632 0 61 9-Monosomy 8; 10-Monosomy 11;40-Monosomy 17; 2-complex Monosomy 8, 11, 17 16633 0 6 2-Monosomy 8;1-Monosomy 11; 3-Monosomy 17 16686 0 55 13-Monosomy 8; 13-Monosomy 11;21 Monosomy 17; 1-Trisomy 8; 1-Trisomy 11; 1-Trisomy 17 16687 0 68612-Monosomy 8; 82-Monosomy 11; 582-Monosomy 17 16720 0 56 8-Monosomy 8;23-Monosomy 11; 25-Monosomy 17 16747 0 58 11-Monosomy 8; 19-Monosomy 11;26-Monosomy 17; 1-Tetrapolid 8; 1-Trisomy 17 16754 0 531 21-Monosomy 8;123-Monosomy 11; 380-Monosomy 17; 7-complex aneuploidy

Although none of the CTCs captured from the blood of Stage III breastcancer patients were stained positive for CK marker (CK+), post-captureanalyses for aneuploidy at chromosome 8, 11 and 17, showed that a largenumber of the captured CTCs are aneuploid cells indicating that theseCTCs are tumor cells. In-situ hybridization study using FISH to detectHer2 (table 4) amplification and aneuploidy (Table 4 and 5) confirmsthat captured CTCs which are CK⁻ are breast cancer cells. The results inTables 4 and 5 show that post-capture molecular analyses, such asamplification of the Her2 marker and detection of aneuploidy of thecaptured cells released from the micro-channel device, positivelyidentify cancer cells in CK⁻ cells from Stage III and IV cancerpatients. This study shows that CTCs captured within the micro-channeldevice provide a robust method for identifying cancer cells which wouldotherwise be left undetected.

Example 12 Post-Capture Molecular Analysis of Captured Cells IncreasesIdentification of CTC as Cancer Cells in Bladder Cancer Patients

Circulating tumor cells (CTCs) were captured from blood samples ofbladder cancer patients. The CTCs were pre-labeled with an antibodycocktail, containing antibodies to CD340, EGFR, CD318, Muc-1, Trop-2,EpCam, Mov-18, MSC, c-met and N-Cadherin. Captured cells were analyzeddirectly within the micro-channel device by fluorescent in-situhybridization for aneuploidy in chromosome 3, 7 and 17, and compared tostaining for CK marker on the captured CTCs.

TABLE 6 Sample #CTCS #Aneuploid Aneuploid Details (Chromosomes ID (CK⁺)cells 3, 7 and 17) 16660 0 17 12-Trisomy 3; 1-Monosomy 3; 2-Monosomy 7;2-Monosomy 17 16664 0 13 1-Trisomy 3; 2-Monosomy 3; 4-Monosomy 7;6-Monosomy 17 16708 0 27 14-Trisomy 3; 2-Monosomy 3; 8-Monosomy 17;2-Monosomy 7; 1-Tetraploid 3 16714 0 78 7-Monosomy 3; 3-Monosomy 7;68-Monosomy 17 16719 0 8 2-Monosomy 3; 1-Monosomy 7; 5-Monosomy 17 167290 29 2-Monosomy 3; 5-Monosomy 7; 10-Monosomy 17; 12-Trisomy 3 16746 0 201-Monosomy 17; 13-Trisomy 3; 1-Trisomy 7; 2-Trisomy 17; 1-Monosomy 3;2-Monosomy 7 16762 0 18 2-Monosomy 3; 12-Monosomy 17; 3-Trisomy 3;1-complex aneuploid (triploid for 3, 7, 17) 16761 0 46 1-Monosomy 3; 5-0Monosomy 7; 8-Monosomy 17; 26-Trisomy 3; 2-Trisomy 17; 4-Tetraploid 3

Table 6 shows that the many of the captured cells from samples obtainedfrom patients with bladder cancer which are stained negative for CK(2^(nd) column) are aneuploid cells (monosomy, trisomy and/or tetraploidat chromosome 3, 7 and 17). The results in Table 6 show that the methodis capable of identifying CTCs from blood obtained from different cancertypes.

The results from these experiments show that the ability to identifyaneuploidy and expression of specific markers in CTCs captured on amicro-channel device provide a means for predicting and managingdiseases, such as cancer during the early stages of tumorigenesis orlate stages of tumorigenesis where tumor cells have metastasized andescaped into the circulation. In addition, the method described is alsoapplicable for monitoring treatment efficacy or failure.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and materials described as these canvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for detecting a target in a sample comprising: contacting asample with a first binding entity to form a pre-loading mixture,wherein the first binding entity specifically binds to a target entityon the surface of the target, passing the pre-loading mixture through amicro-channel, wherein the surface of the micro-channel is coated with asecond binding entity capable of specifically binding, directly orindirectly, to the first binding entity, and detecting the presence ofthe target on the surface of the micro-channel.
 2. The method of claim1, wherein the first binding entity is an antibody or antibody cocktail.3. The method of claim 1, wherein the target is a rare cell in abiological sample.
 4. The method of claim 3, wherein the target cell isa cell present in the biological sample in the ratio of 1 out of 10¹⁰cells, 1 out of 5×10⁷, or 1 out of 10⁴ cells.
 5. The method of claim 3,wherein the target cell is a cancer cell selected from a breast cancercell, a prostate cancer cell, a colorectal cancer cell, a lung cancercell, a pancreatic cancer cell, a ovarian cancer cell, a bladder cancercell, a endometrial cancer cell, a cervical cancer cell, a liver cancercell, a renal cancer cell, a thyroid cancer cell, a bone cancer cell, alymphoma cancer cell, a melanoma cancer cell, and a non-melanoma cancercell.
 6. The method of claim 1, wherein the first binding entityincludes an antibody that specifically binds to an epithelial cellsurface marker.
 7. The method of claim 1, wherein the first bindingentity is an epithelial cell adhesion molecule (EpCAM) antibody.
 8. Themethod of claim 3, wherein (i) the target cell is a breast cancer celland wherein the first binding entity is an antibody that specificallybinds to Her2/neu, EpCAM, MUC-1, EGFR, TAG-12, IGF1R, TACSTD2, CD318,CD104, or N-cadherin, or the first binding entity is an antibodycocktail specifically binding a combination thereof; (ii) the targetcell is a melanoma cancer cell and wherein the first binding entity isan antibody that specifically binds to melanocyte differentiationantigens, oncofetal antigens, tumor specific antigens, SEREX antigens ora combination thereof; (iii) the target cell is a prostate cancer celland wherein the first binding entity is an antibody that specificallybinds to EpCAM, MUC-1, EGFR, PSMA, PSA, TACSTD2, PSCA, PCSA, CD318,CD104, or N-cadherin, or the first binding entity is an antibodycocktail specifically binding a combination thereof; (iv) the targetcell is a colorectal cancer cell and the first binding entity is anantibody that specifically binds to EpCAM, CD66c, CD66e, CEA, TACSTD2,CK20, CD104, MUC-1, CD318, or N-cadherin, or the first binding entity isan antibody cocktail specifically binding a combination thereof; (v) thetarget cell is a lung cancer cell and the first binding entity is anantibody that specifically binds to CK18, CK19, TACSTD2, CD318, CD104,CEA, EGFR, or EpCAM, or the first binding entity is an antibody cocktailspecifically binding a combination thereof; (vi) the target cell is apancreatic cancer cell and the first binding entity is an antibody thatspecifically binds to MUC-1, CEA, TACSTD2, CD104, CD318, N-cadherin,MUC-1, or EpCAM, or the first binding entity is an antibody cocktailspecifically binding a combination thereof; (vii) the target cell is anovarian cancer cell and the first binding entity is an antibody thatspecifically binds to MUC-1, TACSTD2, CEA, CD318, CD104, N-cadherin, orEpCAM, or the first binding entity is an antibody cocktail specificallybinding a combination thereof; (viii) the target cell is an endothelialbladder cancer cell and the first binding entity is an antibody thatspecifically binds to CD34, CD146, CD62, CD105, CD106, VEGF receptor, orMUC-1, or the first binding entity is an antibody cocktail specificallybinding a combination thereof; (ix) the target cell is an epithelialbladder cancer cell and the first binding entity is an antibody thatspecifically binds to TACSTD2, EpCAM, CD318, EGFR, 6B5, N-cadherin orfolate binding receptor, or the first binding entity is an antibodycocktail that specifically binds a combination thereof; (x) the targetcell is a cancer stem cell and the first binding entity is an antibodythat specifically binds to CD133, CD135, CD117, or CD34, or the firstbinding entity is an antibody cocktail that specifically binds acombination thereof; (xi) the target cell is a circulating cancer cellthat expresses mesenchymal antigens and the first binding entity is anantibody that specifically binds to FGFR1, FGFR4, EGFR, folate bindingreceptor, N-cadherin or MSC, or the first binding entity is an antibodythat specifically binds a combination thereof; and/or (xii) the targetcell is a circulating cancer cell that expresses angiogenesis surfaceantigens and the first binding entity is an antibody that specificallybinds to a VEGF receptor.
 9. (canceled)
 10. The method of claim 3,wherein the target cell is a melanoma cancer cell and wherein (i) themelanocyte differentiation antigens consist of tyrosinase, gp75, gp100,MelanA/MART1 or Trp2; (ii) the oncofetal antigens consist of MAGE-A1,MAGE-A4, BAGE, GAGE or NY-ESO1; and/or (iii) the tumor-specific antigensconsist of CDK4 and β-catenin. 11.-22. (canceled)
 23. The method ofclaim 1, wherein the first binding entity is a biotinylated-antibody andthe second binding entity is avidin.
 24. The method of claim 1, furthercomprising contacting the pre-loading mixture with a third bindingentity, wherein the first binding entity is a primary antibody, thethird binding entity is a secondary antibody conjugated to a detectableor capturable entity and specifically binds to the first binding entity,and wherein the second binding entity specifically binds to the thirdbinding entity via the detectable or capturable entity.
 25. The methodof claim 1, further comprises contacting the pre-loading mixture with athird binding entity, wherein the first binding entity is a primaryantibody, the third binding entity is a biotinylated secondary antibodythat specifically binds to the first binding entity, and wherein thesecond binding entity is avidin.
 26. The method of claim 1, wherein thefirst binding entity is a mixture of at least first antibody and secondantibody, and wherein the first antibody specifically binds to a firstepitope of the target entity and the second antibody specifically bindsto a second epitope of the target entity. 27.-35. (canceled)
 36. Adevice comprising: a micro-channel and a loading mixture, wherein themicro-channel comprises a population of posts distributed on the surfaceof the micro-channel in random pattern, wherein the loading mixturecomprises a biological sample suspected of containing a target cell anda first binding entity, wherein the first binding entity specificallybinds to a target entity on the target cell, and wherein the surface ofthe micro-channel is coated with a second binding entity thatspecifically binds, directly or indirectly, to the first binding entity.37. The device of claim 36, wherein the first binding entity comprises abinding entity mixture having at least a first antibody and a secondantibody, and wherein the first antibody specifically binds to a firstepitope of the target entity and the second antibody specifically bindsto a second epitope of the target entity.
 38. The device of claim 36,wherein the biological sample is a physiological fluid or tissueselected from the group consisting of blood, plasma, bone marrow, serum,semen, vaginal secretions, urine, saliva, amniotic fluid, cerebralspinal fluid, synovial fluid, lung lavages, fine needle aspirates (FNA)and biopsy tissue sample.
 39. The device of claim 36, wherein theloading mixture further comprises a third binding entity conjugated to adetectable entity, wherein the first binding entity is a primaryantibody, the third binding entity is a secondary antibody thatspecifically binds to the first binding entity and the second bindingentity specifically binds to the third binding entity.
 40. The device ofclaim 36, wherein the loading mixture further comprises a third bindingentity, wherein the first binding entity is a primary antibody, thethird binding entity is a biotinylated secondary antibody thatspecifically binds to the first binding entity and the second bindingentity is avidin.
 41. (canceled)
 42. A method for capturing a targetcell in a biological sample comprising: contacting a biological samplewith a first binding entity to form a pre-loading mixture, wherein thefirst binding entity specifically binds to a target entity on thesurface of the target cell, passing the pre-loading mixture through amicro-channel, wherein the surface of the micro-channel is coated with asecond binding entity capable of specifically binding to the firstbinding entity, and capturing the target cell on the surface of themicro-channel.
 43. The method of claim 42, wherein the target cell is acirculating tumor cell (CTC). 44.-56. (canceled)